Discovery and development of new antibiotics is a widely recognized, urgent medical need. However, over the past decades, very few new antibiotics have been approved for clinical use and the antibiotic drug pipeline is alarmingly scarce. One of the major factors contributing to the dismal productivity is the lack of technologies that quickly link potent antibacterial compounds to their cellular targets, limiting further development of many compounds. Without a rapid way to make such a link, it is very difficult to advance antibacterial compounds through the lead-development process. Our long-term goal is to discover novel antibiotics employing bacterial functional genomics, molecular biology, bacterial physiology, novel assay development and high throughput approaches. With the current SCORE funding support, we have obtained proof-of-concept for a strategy of target identification using a collection of Escherichia coli cell clones each over-expressing one particular essential gene. In addition, a prototype target identification assay involving 50 cell clones each over-expressing one essential gene was established and validated against three known antibacterial compounds. We expect that a complete over-expression target identification assay will be developed by the end of the current funding period. We propose here to create a complementary target identification technique employing a comprehensive collection of E. coli cell clones each under-expressing one particular essential gene via controlled antisense RNA expression. Specifically, we will construct antisense clones in E. coli cells targeting approximately 250 essential genes and determine optimal induction conditions for sensitization of each cell clone. Subsequently, these antisense clones will be employed to develop a target identification assay which will be validated by using known antibiotics and newly identified antibacterial compounds discovered from a natural product compound library. The E. coli over-expression and under-expression assays, when used in parallel, will provide corroborating as well as complementary results to accelerate the discovery of novel drug candidates with new mechanisms of action to combat multi-drug resistant pathogens.